(F-222) Integrin-Binding Peptide Cooperates with Soluble Transforming Growth Factor Beta 1 to Promote Metastasis in an Engineered Model of Prostate Cancer

Introduction:: Prostate cancer is the most common malignancy detected in men worldwide (1). The tumor microenvironment controls tumorigenesis and hence physiologically relevant biomimetic culture systems presenting essential cell signaling cues have great potential for mechanistic investigation of tumorigenesis and for testing pharmacological inhibitors (2,3). With the aim of modeling prostate cancer metastasis, we engineered a HA-based matrix metalloprotease degradable hydrogel with cell adhesive peptide RGD, that is tunable in a spatial and temporal fashion, by employing the rapid bioorthogonal and extremely efficient cycloaddition reaction between tetrazine (Tz) and trans-cyclooctene (TCO) derivatives (5,7). Epithelial to mesenchymal transition (EMT) is the hallmark of cancer progression and during EMT, upstream signals via growth factors like transforming growth factor beta (TGFβ) leads to activation of transcriptional repressors like Twist, Snail and Zeb1, thereby inhibiting E-cadherin transcription. Loss of cell-cell adhesion proteins contributes to a more invasive phenotype of tumor. Transforming growth factor beta 1 signals were introduced to induce phenotypic changes, modify cell-cell adhesion and cell-matric adhesion, and stimulate cell migration in 3D.


Materials and Methods:: (a) 3D cell culture: PCa cells were suspended in HA-Tz and SMR-bisNb solution to form a homogenous gel. On day 1, RGD-TCO was added to the media to initiate interfacial ligation. On day 3, soluble TGFβ1was added to induce phenotypical changes. Cultures were maintained in EMEM media for 21 days. (b) Immunostaining and confocal imaging: Cell morphology was visualized after 21 days in culture. Cell-laden hydrogel constructs were stained for F-actin using Alexa Fluor 568-phalloidin and nuclei stained with 4’,6-diamidino-2- phenylindole (DAPI) for 2 hours at room temperature. Images were captured using laser scanning microscope 880.


Results, Conclusions, and Discussions:: Results and Discussion: PCa cells encapsulated in the protease degradable HA gels formed multicellular spheroids in 5 days. Incorporation of cell adhesive peptide (RGD, 1 mM) into the hydrogel via a diffusion-controlled interfacial conjugation method caused tumoroid decompaction. Supplementation of the 3D culture with transforming growth factor beta 1 (TGF-β1) induced epithelial-to-mesenchymal transition (EMT), as evidenced by the formation of a dense actin cytoskeleton network (Fig.1) and upregulation of EMT markers and SMAD family related genes.

Conclusion: Overall, matrix-bound RGD peptide cooperates with soluble TGF-β1 to induce a mesenchymal mode of migration, which is reminiscent of tumor metastasis. The tunable 3D cell culture platform can be used to study molecular mechanisms involved in cancer progression and to identify potential molecular targets for cancer therapeutics.


Acknowledgements (Optional): : Acknowledgments: This work was supported by NIH (NIDCR: R01DE029655 and NIGMS: R01GM132460) and NSF (DMR 1809612).


References (Optional): : References: (1)Xu, X, et al, (2012), Biomaterials 33(35), 9049-9060. (2)Hickman, J.A, et al, (2014), Biotechnology Journal 9,1115-1128. (3)Beri, P et al, (2018), Nat Rev Mater 3, 418-430.(4)Hao, Y et al, (2016), Biomacromolecules 17, 3750-3760. (5)Blackman, M.L, et al, (2008), J Am Chem Soc 130, 13518-13519. (6) Peter Friedl et al, (2011), Cell 147. (7)Pol. M, Gao. H, et al, (2022)Biomaterials, under revision.